Bringing it all together: A modern solution for oligonucleotide analysis

In this guest editorial, Joe Fredette, senior business development manager with the Biopharmaceuticals team at Waters, reflects on the recent TIDES meeting - the industry’s largest event for oligonucleotide and peptide therapeutics.

Oligonucleotide therapeutics, mRNA therapies and vaccines dominate the agenda

JF: I was reminded of a blog post I wrote in 2016 after attending my first TIDES meeting: Oligonucleotide Therapeutics on the Rise… Again, July 7, 2016. In his keynote talk, Dr. Muthiah Manoharan, Sr. Vice President for Drug Discovery at Alnylam Pharmaceuticals, provided context around the current state of oligonucleotide-based drug development by referencing the “Gartner Technology Curve,” or “Hype Cycle” – a pattern of development that occurs with many new technologies across a range of industries. He described how the course of oligonucleotide therapeutic development fit this pattern to a T.

In essence, his message was that while oligotherapeutic development had been through a rocky period with drug stability and delivery challenges that needed to be overcome (“The Trough of Disillusionment”), the industry was now poised to enter the next segment of the technology curve: “The Slope of Enlightenment” – a time of sustained growth and advancement. Looking back now over these past six years, how true his prognostication was!

Addressing challenges with stability

JF: Challenges with stability are now being addressed with third and fourth generation chemically modified nucleosides, and drug delivery into targeted cells is being achieved through several different means. From GalNAc (N-Acetylgalactosamine) conjugated oligonucleotides that facilitate uptake by liver cells, to encapsulation by lipid nanoparticles (LNPs) adorned with cell specific ligands, there is no longer uncertainty as to whether nucleic-acid-based drugs can effectively be delivered into targeted cells in-vivo.

Multiple drug approvals in recent years, including the first RNA Interference (RNAi) drugs and the LNP encapsulated mRNA vaccines from Moderna and Pfizer/BioNtech prove they can. Still, there’s room for improvement. Key challenges remain -ensuring the identity and purity of these larger, more complex, multi-component drug formulations is certainly one of them.

Waters advancements

JF: To this end, Waters has long been a leader in developing analytical solutions for oligonucleotides with applications and product development efforts across the analytical value stream; from our technology-leading column chemistries and liquid chromatography (LC) systems to our fit-for-purpose mass spectrometry (MS) tools and compliance-ready informatics.

Solving analytical challenges is at the core of our DNA and when it comes to oligonucleotide analysis, we’re at a point now where we can bring together the many advancements we’ve made across the analytical value stream into complete end-to-end workflows that truly deliver best-in-class analytical performance and productivity.

Separations performance for oligonucleotides

JF: From a liquid chromatography separations standpoint, our new MaxPeak high performance surfaces included with our ACQUITY Premier Systems and LC systems, deliver unparalleled separations performance for oligonucleotides and nucleic acids in particular, given these negatively charged molecules have a strong propensity to adhere to metal surfaces.

New high resolution mass spectrometry products

JF: From a mass spectrometry standpoint, we offer a range of fit-for-purpose MS tools that support everything from in depth characterization to routine monitoring work to QC release testing. In particular, our next generation Xevo G3 QTof platform delivers the mass resolution, sensitivity and flexibility to fully characterize oligonucleotide products and impurities, and our compact BioAccord LC-MS System with SmartMS technology brings the ease-of-use and robustness ideal for GMP monitoring and QC applications in downstream labs (formulation and process development, stability testing, QC release), giving non-MS experts the ability to generate high quality MS data.

Software to accelerate drug development

JF: Tying these technologies together into end-to-end workflows is our compliance-ready waters_connect informatics platform with integrated apps that automate the acquisition, processing and reporting of LC-MS data for a range of applications. With data integrity and compliance-readiness at its core, waters_connect enables a scalable network of high-resolution LC-MS systems that streamlines the deployment and validation of methods across the development, manufacturing, and QC landscape. To learn more about waters_connect informatics platform, I recommend reading the following blog post from another Waters colleague, Rick Boldt, entitled “Connecting Scientists with Next-Generation Informatics” that was published last October.

Waters_connect Informatics Platform

JF: In March of this year, we launched our new waters_connect INTACT Mass app, which automates the mass confirmation and purity analysis of proteins, peptides and oligonucleotides for faster sample analysis, increased data confidence and higher productivity. Along with MaxEnt1, our traditional deconvolution algorithm, it includes the latest monoisotopic BayesSpray deconvolution algorithm, which enhances mass accuracy for molecules in the 1-15kD range, including oligonucleotides. The app also includes a customizable modifications library that enables automated impurities ID and annotation, and it also gives end users the ability to enter custom isotope models that can further enhance performance. To learn more, my colleagues Scott Berger and Patrick Boyce published a great blog post at the time of launch entitled: “Automating Intact Mass Deconvolution: It’s About Time” that describes the INTACT Mass app more fully and puts its development into historical context.

And just earlier this month at the American Society for Mass Spectrometry conference (ASMS 2022) in Minneapolis we were very excited to launch our latest waters_connect informatics platform: CONFIRM Sequence, which automates the sequence confirmation of oligonucleotides and related impurities, including the ID and location of all modifications, and that supports mRNA sequence mapping as well.

New CONFIRM Sequence App

JF: A built-in sequence editor allows end users to build oligonucleotide sequences from scratch or to import them, and an intuitive, menu-driven workflow makes it easy to create and apply automated processing methods. Given a target sequence, the CONFIRM Sequence app automatically generates a list of predicted fragment ions and searches the high energy (MS2) mass spectra for those fragment ions, annotating all matches. The app works with both MS/MS data from the Xevo G3 QTof, or MS^e data from the BioAccord system, and when processing is complete a visual “Dot Map” displays the oligonucleotide sequence and all fragment ions that have been found along with the percent sequence coverage (see below). Interactive links enable a deep dive into the mass spectra of each data point.

Both the INTACT Mass and CONFIRM Sequence apps are the result of a tremendous team effort by Waters software and applications development scientists and engineers, and I’d like to take this opportunity to publicly thank them for the outstanding work they’ve done and are continuing to do. Together, these new apps within waters_connect informatics platform along with the LC and MS tools they acquire data from, represent a modern, scalable solution for oligonucleotide analysis that we’re confident will bring significant value to our customers. Rest assured though; we are not stopping there. We continue to work on new application-based workflows (apps) as well as adding new capabilities to existing apps, and in keeping with our long term vision and “bioharmonization strategy,” what we’re offering today is just the beginning.